Lysosomal membrane permeabilization assay

Giulia Allavena; Doriana Debellis; Roberto Marotta; Chetanchandra S. Joshi; Indira U. Mysorekar; Benedetto Grimaldi

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Lysosomal membrane permeabilization assay

GA Giulia Allavena

DD Doriana Debellis

RM Roberto Marotta

CJ Chetanchandra S. Joshi

IM Indira U. Mysorekar

BG Benedetto Grimaldi

This method is extracted from research article: Cell Death Dis, Jul 2018

A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy

DOI: 10.1038/s41419-018-0786-4

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For lysosomal membrane permeabilization assay, cells were grown on coverslips and treated for 24 h with 10 μM DCAP or 25 μM CQ. A treatment with 2 mM LLoME for 2 h was used as a control³². Samples were then fixed with methanol for 10 min, washed twice with PBS, permeabilized with 0.1% Triton X and blocked with 3% BSA. Cells staining was performed separately with Galectin 1 antibody (1:1000 in BSA 3%, Abcam, ab25138) and SQSTM1 (Santa Cruz, B0316) overnight at 4 °C; matching Alexa Fluor 488- or 555-coupled secondary antibodies (Invitrogen, A11029 and A-21429) were probed 1 h at room temperature with 1:200 dilutions. Images were acquired with A1R+/ A1+ confocal laser microscope system Nikon and quantified using ImageJ software 1.51n (Wayne Rasband, National Institutes of Health).

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